Substrate for Biochip and Method for Manufacturing Substrate for Biochip

ABSTRACT

A substrate for biochips has a substrate surface having a reaction region capable of reacting with biological substances and a non-reaction region not reacting with the biological substances, sunken bottomed wells formed in the substrate surface, and a layer of a material capable of reacting with the biological substances having a surface exposed only at the bottoms of the bottomed wells, the exposed surface forming the reaction region.

TECHNICAL FIELD

The invention relates to a substrate for a biochip, more specifically asubstrate used for making the biochip having biological substances fixedat predetermined positions on the substrate and used for obtaininginformation about various biological samples to be examined. Theinvention relates in particular to the substrate for the biochipsuitable for making a DNA chip by chemically synthesizing probe DNAdirectly on the substrate.

BACKGROUND ART

“Biochip” is the generic term for devices in which biological substancesthat chemically react with to-be-detected biological substances in aspecific manner are fixed at predetermined positions on a chip surface.

A DNA chip that is a typical example of the biochip is used to detectthe types and amounts of target DNA included in blood or cell extract.

The DNA chip has, for example a structure in which thousands to tens ofthousands of types of probe DNA, each being single-chain DNA having aknown sequence, are arranged in an array on a substrate such as a glassslide.

When a to-be-examined liquid containing fluorescence-marked target DNAis supplied to the DNA chip, only the target DNA which has sequencescomplementary to the sequences of the probe DNA is fixed by thecomplementary sequences of target DNA and probe DNA hydrogen-bonding toeach other and forming a double chain. As a result, the parts to whichthe target DNA is fixed is fluorescent-colored. By measuring theposition and coloring intensity of the fluorescent-colored parts on thechip, the types and amounts of the target DNA can be detected.

In order to make a DNA chip used in this manner, it is necessary to fixa plurality types of probe DNA each having a predetermined sequence atpredetermined positions on a substrate surface.

There are two main methods of fixing probe DNA. A first method is called“micro array method”. In this method, probe DNA that has been chemicallysynthesized or extracted from an intended living organism in advance isfixed in an array on a substrate by dropping or printing.

In a second method, using four bases thymine (T), adenine (A), cytosine(C) and guanine (G), a plurality of types of probe DNA, each beingsingle-chain DNA having a predetermined sequence according to design,are chemically synthesized directly on a substrate.

In the second fixing method, it is required that on a surface of asubstrate used for the chemical synthesis, a reaction region where areaction to synthesize intended probe DNA proceeds and a non-reactionregion which is not involved in the synthesis reaction should be formedin a definitely separated manner.

As a DNA chip made by this second fixing method, for example “Gene Chip”(name of an article produced by Affymetrix, Inc.) is known.

In the case of this chip, quartz is used as a material for a substrate,and photolithography is applied to form a reaction region and anon-reaction reaction in a separated manner. Further, when probe DNA ischemically synthesized, single-chain DNA is formed unit by unit byapplying ultraviolet rays for activation of single-chain DNA in thereaction region.

In this chip, a reaction region consisting of about 200,000 spots ofabout 20 μm square each is formed on a single substrate, and about2,000,000 strands of probe DNA of the same type are fixed in one spot.

In this chip, the spots are formed on the chip at high density. Hence, avery large number of types of target DNA can be detected in one test.

FIG. 2A of PCT Application Published Japanese Translation No. Hei9-500568 shows an array plate described below.

The array plate is made as follows: First, a surface of an Si substrateis made to react with fluoroalkylsilane to once form a thin film ofhydrophobic fluoroalkylsiloxane on it. Then, this thin film is removedin a predetermined two-dimensional pattern so that the surface of the Sisubstrate will be exposed in the spots having the thin film removed.Last, the exposed surface is made to react with hydroxysilane oralkylsilane so that the exposed surface will have OH group.

Thus, in this array plate, the surface of the Si substrate has sites ofa hydrophobic thin film having a large surface tension and sites havinghydrophilic OH group. For biological substances, the former function asa non-reaction region, while the latter function as a reaction region.

When this array plate is used, synthesis reaction is made to proceed inthe hydrophilic sites, and then a to-be-examined liquid is supplied tothese sites. Due to the large surface tension of the hydrophobic thinfilm around the hydrophilic sites, the to-be-examined liquid is held inthe hydrophilic sites.

In this array plate, however, the reaction region and the non-reactionregion are formed on the surface of the Si substrate to be virtuallyflush with each other. Hence it does not have sufficient stability inholding a supplied to-be-examined liquid and hence is not easy to use.

Further, the formation of the reaction region and the formation of thenon-reaction region both depend on chemical reaction between the Sisurface and other chemicals. The reaction does not always proceed at ayield of 100%. Thus, it is not improbable that the boundary between thereaction region and the non-reaction region is indefinite. Anotherproblem is that the hydrophobic thin film and the hydrophilic sites areeasily damaged from the outside.

From the aspect of holding an to-be-examined liquid, a substrate inwhich bottomed wells having a structure capable of holding ato-be-examined liquid are distributed in a substrate surface ispreferable to the array plate having the above structure.

As a substrate of this type, PCT Application Published JapaneseTranslation No. 2002-537869 discloses a substrate used for directsynthesis of probe DNA and a method of chemical synthesis of probe DNAusing it.

A sketch of this substrate is given in FIG. 1.

The substrate 1 is made from an Si wafer. In a surface of the substrate1, a plurality of micro cuvettes 2 are formed in a predetermined array.The micro cuvettes 2 are sunken holes (bottomed wells) of about 1 to1000 μm in diameter and about 1 to 500 μm in depth, and function as areaction region for chemical synthesis of probe DNA. The surface partexcept for these wells is a non-reaction region.

This substrate A is made as follows:

Step a₁: By applying photolithography and etching to a surface 1 _(a) ofan Si wafer 1, an intermediate A₁ as shown in FIG. 2 is formed, in whichsunken holes 2A of almost the same shape as that of to-be-formedbottomed wells are formed at positions at which the bottomed wells areto be formed.

Step a₂: By performing, for example thermal oxidation treatment onto thesurface 1 _(a) of the Si wafer and the surfaces of the sunken holes 2A(their bottoms and side surfaces) in the intermediate A₁, anintermediate A₂ as shown in FIG. 3 is formed, in which only thesuperficial part of these surfaces has been turned into an Si oxidelayer 3 of about 0.5 μm in thickness.

Step a₃: Silanization treatment is performed onto the surface of the Sioxide layer 3 of the intermediate A₂. Specifically, the surface of theSi oxide layer 3 is treated with alkali by applying Brown Process usingNaOH, and then treated with, for example an activated silanizing agenthaving an epoxysilane type group. Then, linkage of the silanizing agentand hydrolysis of epoxy resin is made to proceed successively. As aresult, an intermediate A₃ shown in FIG. 4 is obtained, in which asilane layer 4 is formed on the surface of the Si oxide layer 3.

In the intermediate A₃, since OH groups of silane are present in itsentire surface, the entire surface can react with DNA phosphoramidite.

Step a₄: By applying the phosphoramidite method to the surface of theintermediate A₃, strands of single-chain DNA of a length correspondingto 5 bases or so are synthesized using DNA phosphoramidite T (thymine).Thus, an intermediate A₄ having bottomed wells as shown in FIG. 5 isformed, in which an oligonucleotide (5T) spacer layer 5 is formed on thesilane layer 4.

It is to be noted that the terminal of the synthesized oligonucleotide(5T) is blocked with dimethoxytrityl (DMT).

The entire surface of the intermediate A₄ is covered with the spacerlayer 5 of oligonucleotides (5T) blocked with DMT. Hence, when theterminals of oligonucleotides are activated by eliminating the DMTblocking the terminals (detrirylation), probe DNA can be synthesizedthere.

Thus, in the case of the intermediate A₄, when a DNA chip is to be made,the entire surface formed of the oligonucleotide (5T) spacer layer 5functions as a reaction region.

However, this does not meet the condition for the substrate forsynthesizing probe DNA, namely the condition that the bottomed wells andthe other part should be definitely separated as a reaction region and anon-reaction region, respectively.

Hence, treatment needs to be performed on the layer 5 of theintermediate A₄ to leave the bottomed wells as they are a reactionregion and turn the other part into a non-reaction region. Thistreatment is capping performed in the next step.

Step a₅: As shown in FIG. 6, in the intermediate A₄, only the bottomedwells are filled with resin droplets 6. In this state, capping isperformed on the layer 5.

Specifically, by performing detritylation on the oligonucleotide (5T)spacer layer 5, the terminals of the oligonucleotides (5T) areactivated. Then, using trichloroacetic acid, acetic anhydride,dymethylaminopyridine or the like, the activated terminals of theoligonucleotides (5T) are blocked and inactivated.

Then, using an organic solvent such as tetrahydrofuran, the resindroplets 6 filling the bottomed wells are dissolved and removed so thatthe layer 5 will be exposed in the bottomed wells.

During this capping, since the bottomed wells are filled with resin, theoligonucleotide (5T) spacer layer 5 in the bottomed wells does notundergo the capping and maintains the state capable of reaction.Meanwhile, the oligonucleotides (5T) in the part except for the bottomedwells undergo the capping and are brought into a state incapable ofsynthesis reaction.

In this way, a substrate A for synthesis of probe DNA having across-sectional structure shown in FIG. 7 is made.

In this substrate A, the bottomed wells 2, which are sunken holes, areformed in the surface of the Si wafer 1 in a predetermined pattern. TheSi oxide layer 3 is formed on the Si wafer 1 to cover the entire surfacethereof, and the silane layer 4 is formed on the Si oxide layer 3 tocover the entire surface thereof.

On the bottom 2 a and the side surface 2 b of each of the bottomed wells2, the spacer layer 5 of oligonucleotides (5T) with their terminalsblocked with DMT is exposed. These spots form a reaction region forsynthesizing probe DNA. Meanwhile, the part 5 a except for these spotsof the layer 5 has undergone capping and forms a non-reaction region.

When a DNA chip is made using this substrate A by the phosphoramiditemethod, chemical synthesis of probe DNA proceeds in the bottomed wells2.

However, this substrate A has problems mentioned below.

A first problem is that in the substrate A, the boundary between thereaction region and the non-reaction region is determined by how thebottomed wells are filled with resin droplets in step a₅.

Generally, filling of the bottomed wells with resin droplets isperformed using a piezoinjector. The amount of resin droplets suppliedto fill one bottomed well is very minute, specifically in the order ofp1 to μ1. Hence, in step a₅, sometimes the amount of resin dropletssupplied to fill a bottomed well is too much and the resin runs over thebottomed well, and sometimes the amount of resin droplets supplied istoo less to completely fill a bottomed well.

In the former case, also a surface of a part surrounding the bottomedwell is covered with the resin running over the bottomed well. As aresult, in the capping performed next, the surface of this surroundingpart does not undergo capping and remains capable of synthesis reaction.

Hence, when a DNA chip is made, probe DNA is chemically synthesized alsoon the surface of the part surrounding the bottomed well. As a result,when target DNA is examined using the DNA chip made, fluorescentcoloring may occur not only in the bottomed well but also in the partsurrounding the bottomed well over which the resin ran. This hindersaccurate reading of fluorescent marks.

Further, in the case in which the amount of resin droplets supplied istoo less to fill a bottomed well, the thickness of the resin coveringthe inside surface of the bottomed well is thin. Hence, the resin iseasily corroded by an acid solution used in capping. As a result, a partof the oligonucleotide (5T) spacer layer located in the bottomed wellmay undergo capping.

Thus, when a DNA chip is made, probe DNA may not be satisfactorilychemically synthesized in this bottomed well. As a result, when targetDNA is examined, fluorescent coloring may not occur with a sufficientcoloring intensity in this bottomed well.

Further, if the capping in step a₅ is insufficient, it also may causethe problem that when a DNA chip is made and used, fluorescent coloringoccurs not only in bottomed wells but also in parts where capping wasinsufficient. In other words, background noise easily occurs.

Further, when the sunken holes 2A, which will form the bottomed wells,are formed in step a₁, the depth of the sunken holes 2A is determined bythe length of etching time. Hence, if time management for etching is notperformed accurately, the bottomed wells may not be formed to have anaccurate depth according to design criteria.

DISCLOSURE OF INVENTION

An object of the invention is to provide a substrate for a biochip whichcan be made without capping that is an indispensable step (step a₅) formaking the substrate A described as an example of the substrate for thebiochip having bottomed wells, and still in which the boundary between areaction region and a non-reaction region is definite.

Another object of the invention is to provide a substrate for a biochipin which bottomed wells having an accurate depth according to designcriteria can be much more easily formed than in conventional substrates.

In order to achieve the above objects, the invention provides asubstrate for a biochip, comprising a substrate surface having areaction region capable of reacting with biological substances and anon-reaction region not reacting with the biological substances, sunkenbottomed wells formed in the substrate surface, and a layer of amaterial capable of reacting the biological substances having a surfaceexposed only at the bottoms of the bottomed wells, the exposed surfaceforming the reaction region.

In particular, the invention provides the substrate for the biochip inwhich the layer of a material capable of reacting the biologicalsubstances is a layer of a silicon (Si) oxide, and in which the surfaceof the Si oxide layer is silanized.

Yet another aspect of the present invention inheres in a substrate for abiochip according to the embodiment of the present invention having afirst layer having a surface to be hydroxylated, and a second layerdisposed on the first layer, the second layer having a plurality ofwells reaching to the first layer and a plurality of grooves configuredto fill the wells with a same solution.

Yet another aspect of the present invention inheres in a method formanufacturing a substrate for a biochip according to the embodiment ofthe present invention having etching portions of a second layer disposedon a first layer and exhibiting the first layer, and dipping the firstlayer into a sodium hydrate solution to introduce a plurality ofhydroxyl groups on the first layer.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a perspective view showing an example of a substrate forsynthesizing probe DNA;

FIG. 2 is a cross-sectional view showing an intermediate A₁ in theformation of a conventional substrate A for synthesizing probe DNA;

FIG. 3 is a cross-sectional view showing an intermediate A₂ in theformation of the substrate A;

FIG. 4 is a cross-sectional view showing an intermediate A₃ in theformation of the substrate A;

FIG. 5 is a cross-sectional view showing an intermediate A₄ in theformation of the substrate A;

FIG. 6 is a cross-sectional view showing an intermediate A₅ in theformation of the substrate A;

FIG. 7 is a cross-sectional view showing an example A of a conventionalsubstrate for synthesizing probe DNA;

FIG. 8 is a cross-sectional view showing an example B₀ of substratestructure for a substrate according to a first embodiment of the presentinvention;

FIG. 9 is a cross-sectional view showing an example B₁ of a substratefor synthesizing prove DNA according to the first embodiment of thepresent invention;

FIG. 10 is a cross-sectional view showing a starting material used forforming the substrate B₁:

FIG. 11 is a cross-sectional view showing the material of FIG. 10 with aresist formed on, a surface thereof;

FIG. 12 is a cross-sectional view showing the material of FIG. 11 inwhich openings are formed in the resist;

FIG. 13 is a cross-sectional view showing the material in which holesfor wells are formed to have a surface of an Si oxide layer exposed;

FIG. 14 is a cross-sectional view showing the material in which ansilane layer is formed on the surface of the Si oxide layer;

FIG. 15 is a cross-sectional view showing another substrate B₂ accordingto the first embodiment of the present invention;

FIG. 16 is a cross-sectional view showing another substrate B₃ accordingto the first embodiment of the present invention;

FIG. 17 is a cross-sectional view showing another substrate B₄ accordingto the first embodiment of the present invention;

FIG. 18 is an explanatory cross-sectional diagram showing a structure ofthe substrate B₁;

FIG. 19 is an explanatory cross-sectional diagram showing the substrateB₁, where a well is filled with resin droplet to mask oligonucleotides(5T) space layer;

FIG. 20 is an explanatory cross-sectional diagram showing the substrateB₁, where DMT has been eliminated from the terminals of oligonucleotides(5T) space layer in an open bottomed well;

FIG. 21 is an explanatory cross-sectional diagram showing the substrateB₁, where the resin that had filled the bottomed well has been dissolvedand removed;

FIG. 22 is an explanatory cross-sectional diagram showing the substrateB₁, where DNA phosphoramidites (C) is chemically bonded to theoligonucleotides (5T) from which DMT has been eliminated;

FIG. 23 is a photograph showing results of examination about target DNAusing a DNA chip made using the substrate B₁ according to the firstembodiment of the present invention;

FIG. 24 is a photograph showing results of examination about target DNAusing a DNA chip made using the conventional substrate A;

FIG. 25 is a plane view of a substrate for a biochip in accordance witha third embodiment of the present invention;

FIG. 26 is a first sectional view taken along line XXVI-XXVI in FIG. 25,showing the substrate for the biochip in accordance with the thirdembodiment of the present invention;

FIG. 27 is a sectional view taken along line XXVII-XXVII in FIG. 25,showing the substrate for the biochip in accordance with the thirdembodiment of the present invention;

FIG. 28 is an enlarged sectional view of a biomolecule layer inaccordance with the third embodiment of the present invention;

FIG. 29 is a second sectional view taken along line XXVI-XXVI in FIG.25, showing the substrate for the biochip in accordance with the thirdembodiment of the present invention;

FIG. 30 is a first enlarged sectional view of a silane film and thebiomolecule layer in accordance with the third embodiment of the presentinvention;

FIG. 31 is a second enlarged sectional view of the silane film and thebiomolecule layer in accordance with the third embodiment of the presentinvention;

FIG. 32 is a plane view of a cover plate in accordance with the thirdembodiment of the present invention;

FIG. 33 is a sectional view taken along line XXXIII-XXXIII in FIG. 32,showing the cover plate in accordance with the third embodiment of thepresent invention;

FIG. 34 is a sectional view of the cover plate disposed on the substratefor the biochip in accordance with the third embodiment of the presentinvention;

FIG. 35 is a first sectional view of the substrate for the biochipdepicting a manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 36 is a second sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 37 is a third sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 38 is a plane view of the substrate for the biochip depicting themanufacturing process in accordance with the third embodiment of thepresent invention;

FIG. 39 is a fourth sectional view taken along line XXXIX-XXXIX in FIG.38, depicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 40 is a fifth sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 41 is a sixth sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 42 is a seventh sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 43 is a eighth sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 44 is a ninth sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 45 is a tenth sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 46 is a eleventh sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 47 depicts protected bases in accordance with the third embodimentof the present invention;

FIG. 48 is a twelfth sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 49 is a thirteenth sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 50 depicts a phosphoramidite in accordance with the thirdembodiment of the present invention;

FIG. 51 is a fourteenth sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 52 is a fifteenth sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 53 is a sixteenth sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 54 is a seventeenth sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 55 depicts deprotected bases in accordance with the thirdembodiment of the present invention;

FIG. 56 is an eighteenth sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the thirdembodiment of the present invention;

FIG. 57( a) shows a fluorescent image on the substrate for the biochipin accordance with the third embodiment of the present invention, andFIG. 57( b) shows a fluorescent image on an earlier substrate for thebiochip;

FIG. 58 is a first sectional view of a substrate for the biochipdepicting a manufacturing process in accordance with a firstmodification of the third embodiment of the present invention;

FIG. 59 is a second sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the firstmodification of the third embodiment of the present invention;

FIG. 60 is a third sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the firstmodification of the third embodiment of the present invention;

FIG. 61 is a fourth sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the firstmodification of the third embodiment of the present invention;

FIG. 62 is a fifth sectional view of the substrate for the biochipdepicting the manufacturing process in accordance with the firstmodification of the third embodiment of the present invention;

FIG. 63 is a plane view of a substrate for the biochip in accordancewith a second modification of the third embodiment of the presentinvention;

FIG. 64 is a first sectional view taken along line LXIV-LXIV in FIG. 63,showing the substrate for the biochip in accordance with the secondmodification of the third embodiment of the present invention;

FIG. 65 is a plane view of a cover plate in accordance with the secondmodification of the third embodiment of the present invention;

FIG. 66 is a sectional view taken along line LXVI-LXVI in FIG. 65,showing the cover plate in accordance with the second modification ofthe third embodiment of the present invention; and

FIG. 67 is a sectional view of the cover plate on the substrate for thebiochip in accordance with the second modification of the thirdembodiment of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

In a substrate for a biochip according to the invention, a reactionregion capable of reacting with biological substances and a non-reactionregion not reacting with the biological substances are formed on asubstrate surface in a definitely separated manner.

Here, the term “biological substances” is used to mean biologicalmolecules or physiologically active substances having a site reactingwith or bonding to-be-examined substances. Further, the term “biologicalsubstances” is used here to include substances that have an affinitywith the biological substances in the above sense. The biologicalsubstances are not limited to specific ones. For example, substanceshaving a site bonding to nucleic acids such as oligonucleotide,polynucleotide, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA);substances having a physiologically or pharmacologically active sitesuch as enzymes, vitamins, peptides, proteins, hormones, endocrinedisturbing chemicals, sugars and lipids; complexes of RNA and a proteinand complexes of a protein such as lectin and a sugar can be mentioned.

By fixing biological substances that react with to-be-examinedbiological substances in a specific manner in a reaction region of thesubstrate for the biochip according to the invention, different types ofchips can be made.

Using a substrate for synthesizing probe DNA as an example, thesubstrate for the biochip according to the invention will be describedbelow.

FIRST EMBODIMENT

The substrate according to a first embodiment of the present inventionis similar to the above-described substrate A of FIG. 7 in that asurface of the substrate comprises a reaction region where reaction inwhich probe DNA is formed using DNA phosphoramidite can proceed and anon-reaction region where this reaction does not proceed.

However, in the substrate A, the non-reaction region is formed bycapping. Meanwhile, in the substrate according to the first embodimentof the present invention, the reaction region is formed of a reactivematerial while the other region is surely formed of a material inactivein the reaction. Thus, the most important feature of the substrateaccording to the first embodiment of the present invention is that thereaction region and the non-reaction region are formed in a definitelyseparated manner without performing surface inactivation treatment suchas capping.

First, an example B₀ of the basic structure for the substrate accordingto the first embodiment of the present invention is shown in FIG. 8.

The substrate B₀ is a plate-like piece having a sandwich structurecomprising two Si layers 131, 131 and an Si oxide layer 132 providedbetween them, where holes 13A reaching to the surface 132 a of the Sioxide layer 132 are formed in one of the Si layers 131 in apredetermined array pattern to function as bottomed wells.

Thus, in the substrate B₀, the surface 132 a of the Si oxide layer 132is exposed only at the bottoms of the holes 13A, and the other surfaceof the substrate B₀ is in Si. Only the surface of the Si oxide layer 132functions as a reaction region for synthesizing, for examplesingle-chain DNA, while all the other surface of the substrate B₀functions as a non-reaction region.

FIG. 9 shows another example B₁ of a substrate, which is made using theabove-described substrate B₀.

In this substrate B₁, a silane layer 14 is formed only at the bottoms ofthe holes 13A in the substrate B₀. On the silane layer 14 is formed aspacer layer 135 of oligonucleotides (5T) each formed by synthesizing 5units of DNA phosphoramidite T successively. As a result, the holes 13Afunction as bottomed wells 13.

In this substrate B₁, the oligonucleotide (5T) spacer layer 135 whichenables synthesis of probe DNA is exposed only at the bottoms 135 a ofthe bottomed wells 13, while the side surfaces 135 b of the bottomedwells 13 and the surface 131 a of the Si layer 131 remain in Si.

Hence, in the substrate B₁, only the bottoms of the bottomed wells 13function as a reaction region for synthesizing probe DNA, while theother part including the side surfaces of the bottomed wells 13 and thesubstrate surface is a non-reaction region in the chemical synthesis ofprobe DNA, as it is, namely without undergoing surface inactivationtreatment such as capping.

It is to be noted that the silane layer 14 in the substrate B₁ isprovided so that the oligonucleotide (5T) spacer layer 135 can besynthesized efficiently, and is not indispensable, because theoligonucleotide (5T) spacer layer 135 can be synthesized directly on thesurface 132 a of the Si oxide layer 132.

The substrate B₁ can be made as follows:

First, as shown in FIG. 10, a plate-like piece of a structure in whichan Si oxide layer 132 is sandwiched between two Si layers 131, 131 isprepared. It is to be noted that one of the Si layers 131 should have athickness almost equal to the depth of to-be-formed bottomed wells.

As the plate-like piece of this type, an SOI (Silicon on Insulator)wafer commercially available from Shin-Etsu Chemical Co., Ltd. issuitable.

Next, as shown in FIG. 11, the entire surface 131 a of one of the Silayers 131 is covered with a resist 16, a mask having openings of thesame diameter as that of the to-be-formed bottomed wells is placed onthe resist, and ultraviolet rays are applied. Then, the mask is removedand the whole is developed.

As a result, as shown in FIG. 12, openings of the same diameter as thatof the to-be-formed bottomed wells are formed in the resist 16, and thesurface 131 a of the Si layer 131 is exposed in these openings.

Then, using the resist 16 as a mask, etching is performed on the Silayer 131 using an Si etchant to form holes 13A reaching to the surface132 a of the Si oxide layer 132.

The etching automatically stops at the time the removal of the Si layerreaches to the surface 132 a of the Si oxide layer 132.

As a result, as shown in FIG. 13, the piece in which the surface 132 aof the Si oxide layer 132 is exposed only at the bottoms of the holes13A and the other surface part is made of Si is obtained. It is to benoted that the holes 13A in this piece have almost the same diameter anddepth as those of the to-be-formed bottomed wells.

Then, silanization treatment is performed. The silanization reactionproceeds on the surface of the Si oxide layer, so that a silane layer 14is formed only at the bottoms 132 a of the holes 13A (FIG. 14).

Last, by applying the phosphoramidite method, the silan layer 14 is madeto react with DNA phosphoramidite T to form a spacer layer 135 ofoligonucleotides (5T) each being a strand of sing-chain DNA of 5 mer T.Thus, the substrate B₁ shown in FIG. 9 is obtained.

In this process, DNA phosphoramidite forms covalent bonding only withOH-group in the silance layer. Hence, the synthesis reaction proceedsonly at the bottoms 132 a of the bottomed wells. Meanwhile, since DNAphosphoramidite T does not react with the part where Si is exposed, thispart remains a non-reaction region although capping is not performed,unlike the case with the substrate A.

The comparison between the process of making the substrate B₁ (B₀) andthe process of making the conventional substrate A reveals thefollowing:

First, in the case of the substrate A, in step a₂, it is necessary toperform thermal oxidation treatment on the surface of the Si wafer toform an Si oxide layer. Meanwhile, the substrate B₁ (B₀) according tothe first embodiment of the present invention does not require step a₂.

Further, in the case of the substrate A, in step a₅, a series ofoperations, namely filling the bottomed wells with resin droplets,capping, and dissolution and removal of the resin are indispensable.Meanwhile, the substrate B₁ according to the first embodiment of thepresent invention does not require these operations at all.

Hence, in the case of the substrate B₁ (B₀), the problems caused bysupplying the bottomed wells with too much or too less an amount ofresin droplets are obviated, and the boundary between the reactionregion and the non-reaction region is definite. Further, the reactionregion consists only of the bottoms of the holes 13A formed by applyingphotolithography and etching, and the other surface part forms thenon-reaction region. Thus, when a DNA chip made using this substrate isused, it allows high-accuracy reading of fluorescent marks and preventsbackground noise.

Further, in the case of the substrate A, in order to form the bottomedwells to have a depth according to design criteria, it is necessary toaccurately manage etching time for etching in step a₁. Meanwhile, in thecase of the substrate B₁ (B₀) etching which removes parts of the Silayer 131 automatically stops at the time the removal reaches to thesurface of the Si oxide layer. The depth of the holes formed isdetermined uniquely by the thickness of the Si layer 131 used. Hence,the bottomed wells formed have a very accurate depth.

Another example B₂ of a substrate is shown in FIG. 15.

In the substrate B₂, on one side of an Si plate 141, only a superficialpart is turned into an Si oxide layer 141 a, for example by thermaloxidation. On this Si oxide layer 141 a is formed a layer 142 of amaterial which does not react with biological substances such as DNAphosphoramidite T and is not corroded by organic solvents, acidsolutions, alkali solutions or the like used in synthesis of DNA, in apredetermined thickness. Across the thickness of this layer 142, holes23 of a predetermined diameter are formed up to the surface 141 b of theSi oxide layer 141 a, in a predetermined array pattern.

On the surface 141 b of the Si oxide layer 141 a, a silane layer 14 anda spacer layer 135 of oligonucleotides (5T), each being a strand of 5mer single-chain DNA formed from reaction between the silane layer 14and DNA phosphoramidite T, are formed in this order.

In the case of the substrate B₂, the surface of the oligonucleotide (5T)spacer layer 135 capable of reacting with DNA phosphoramidite T isexposed only at the bottoms of the holes 23 to form a reaction region.All the other surface part is a non-reaction region made of a materialnot reacting with DNA phosphoramidite T, although it does not undergoinactivation treatment.

Here, as a material for the layer 142 which forms a non-reaction region,for example single-crystal silicon, metals not easily forming an oxidesuch as platinum, nitrides such as silicon nitride, plastics not havinga reactive functional group such as polyethylene and polystyrene can bementioned. The layer 142 can be formed by a film formation method suchas direct joint of the material as mentioned above to the Si oxidelayer, vacuum deposition or CVD of the material as mentioned above ontothe Si oxide layer surface, vapor phase polymerization of a monomer. Theholes 23 can be formed by application of photolithography and etching,for example.

Another example B₃ of a substrate is shown in FIG. 16.

In the substrate B₃, the same layer 142 as that of the substrate B₂ isformed on a surface of a glass plate 151. Across the thickness of thislayer 142, holes 152 reaching to the surface 151 _(a) of the glass plate151 are formed as bottomed wells, and only on the surface 151 _(a) ofthe glass plate exposed at the bottoms of the holes 152, a silane layer14 and an oligonucleotide (5T) spacer layer 135 are formed.

In the case of the substrate B₃, the surface of the oligonucleotide (5T)spacer layer is exposed only at the bottoms 151 _(a) of the holes 152 toform a reaction region. The side surfaces of the bottomed wells and thesurface of the layer 142 form a non-reaction region although they do notundergo inactivation treatment.

Another example B₄ of a substrate is shown in FIG. 17.

In the substrate B₄, holes of a predetermined diameter and depth areformed in a surface 161 _(a) of a plate-like piece 161 made of a similarkind of material which does not react with DNA phosphoramidite T and isnot corroded by organic solvents, acid solutions, alkali solutions orthe like used in synthesis of DNA. These holes are made into bottomedwells by forming an Si oxide layer 163 at the bottoms 162 _(a) of theholes, and then forming a silane layer 14 and an oligonucleotide (5T)spacer on the Si oxide layer 163 in this order.

Also in the case of the substrate B₄, the oligonucleotide (5T) spacerlayer located 135 at the bottoms of the bottomed wells is only the partthat can react with DNA phosphoramidite T, while the other part is anon-reaction region.

As the material forming the non-reaction region, Si and nitrides such assilicon nitride can be mentioned, for example.

When a plate-like piece of Si is used, the above-mentioned bottomedwells can be formed by applying dry etching such as reactive ion etchingor ion milling, or wet etching, for example.

The bottomed wells may be formed as follows: By applying the SIMOX(Separation by Implanted Oxygen) method to put in oxygen ions from asurface of the Si plate, an Si oxide layer is formed at a certain depthfrom the surface of the Si plate. Then, by forming holes from thesurface of the Si plate up to the Si oxide layer, the bottomed wells areobtained.

Alternatively, the bottomed wells may be formed as follows: A surface ofthe Si plate is once oxidized. Then by applying photolithography andetching to the resulting Si oxide surface layer, openings are formed ina predetermined array pattern. Then, oxygen ions are put in from thesurface of Si exposed in the openings by the above-mentioned SIMOXmethod, and then the Si oxide surface layer is removed by etching. As aresult, the structure in which an Si oxide layer extending only justunder the places that were the openings is formed at a certain depthfrom the surface of the Si plate is obtained. Then, by forming holesfrom the surface of the Si plate up to the Si oxide layer in the placesthat were the openings, the bottomed wells are obtained.

Next, the process of making a biochip using the substrate for thebiochip according to the first embodiment of the present invention willbe described using an example in which a DNA chip having probe DNA fixedby the phosphoramidite method is made using the substrate B₁ shown inFIG. 9.

First, the substrate B₁ is prepared. As shown in a simplified manner inFIG. 18, in each of the bottomed wells 13 that are arranged in an arrayin the substrate B₁, oligonucleotides (T5) space layer 135 having theirterminals blocked with DMT are fixed only at the bottom of the bottomedwell.

Step b₁: Resin masking is performed for the bottomed wells.Specifically, of the bottomed wells arranged, all the bottomed wellsexcept for those which should undergo chemical reaction with DNAphosphoramidite C (cytosine), for example are filled with resin droplets6.

As shown in FIG. 19, the oligonucleotides (5T) space layer in thebottomed well filled with resin droplet 6 are blocked by the resin,while the oligonucleotides (5T) space layer in the bottomed well notfilled with resin droplet 6 are in a state that DMT at their terminalscan be eliminated.

Step b₂: Detritilation is performed by supplying a solution of an acidsuch as trichloroacetic acid evenly to the entire upper surface of thesubstrate.

As a result, as shown in FIG. 20, in the bottomed wells not filled withresin droplets 6, DMT is eliminated from the oligonucleotides (5T) spacelayer, so that those oligonucleotides (5T) space layer are activated.

Thus, of the bottomed wells arranged in the surface of the substrate B₁,only the specific wells (wells not filled with resin droplets) are in astate capable of reacting with DNA phosphoramidite.

Step b₃: By supplying an organic solvent to the substrate surface, theresin droplet filling the bottomed wells is dissolved and removed.

As a result, as shown in FIG. 21, at the bottoms of the bottomed wellsarranged in the substrate, the activated oligonucleotides (5T) spacelayer and the oligonucleotides (5T) space layer having their terminalsblocked with DMT are exposed.

Step b₄: DNA coupling is performed by supplying a reagent containing DNAphosphoramidites C with their terminals blocked with DMT evenly to theentire surface of the substrate.

As a result, as shown in FIG. 22, in the specific bottomed wells,synthesis reaction between the activated oligonucleotides (5T) spacelayer and the supplied DNA phosphoramidites C proceed, so that they arechemically combined together. Thus, the oligonucleotides grow by 1 mer,and the terminals of the grown oligonucleotides are blocked with DMT.

Meanwhile, in the other bottomed wells, the exposed oligonucleotides(5T) space layer have their terminals blocked with DMT and therefore areinactive. Hence, they do not react with the supplied DNAphosphoramidites C.

By performing this DNA synthesis reaction where steps b₁ to b₄ form onecycle, only in the bottomed wells not filled with resin droplets, theoligonucleotides grow by 1 mer in one cycle. Meanwhile, the bottomedwells filled with resin remains as they were before the synthesisreaction started.

There are four types of DNA phosphoramidite T, A, C and G to react withthe oligonucleotides. Hence, by performing the above cycle four times,the oligonucleotides fixed in the bottomed wells can be grown by 1 mer.

In this way, probe DNA having sequences according to a design can befixed only at the bottoms of the bottomed wells in the substrate B₁.

When a DNA chip is made using this substrate B₁, DNA synthesis reactionproceeds only at the bottoms of the bottomed wells. The other part isnot involved in the synthesis reaction at all. Hence, for example evenif in step b₁, resin droplet to fill a bottomed well runs over thebottomed well, it does not affect the DNA synthesis reaction at allunless the resin flows into adjacent bottomed wells, and therefore hasno adverse effects on reading of fluorescent marks at all. Further,inactivation treatment (capping) which is performed in the case of thesubstrate A does not need to be performed any longer.

In the above description, all the substrates B₁, B₂, B₃ and B₄ aresubstrates for DNA chips in which the formation of the silane layer onthe surface of the Si oxide layer of the substrate B₀ enables probe DNAsynthesis reaction.

However, the substrate for the biochip according to the first embodimentof the present invention is not limited to this type. By selecting, as amaterial for forming the bottoms of the bottomed wells (reactionregion), a material capable of fixing biological substances that bond toto-be-examined biological substances in a specific manner, differenttypes of chips can be made.

For example, if a linker substance that bonds to a to-be-examinedbiological substance in a specific manner is fixed on the surface of theSi oxide layer of the substrate B₀ having the basic structure shown inFIG. 8, the part to which the linker substance is fixed can function asa reaction region specialized for reaction with that biologicalsubstance. In this case, the other part of the substrate forms anon-reaction region for that biological substance, and both regions areseparated by a definite boundary.

For example when a silane coupling agent such asaminopropylemethoxysilane, or a substance having a functional group suchas an epoxy group, an tosyl group, an activated carboxyl group, an aminogroup, a thiol group or a bromoatoamido group is used as the linkersubstance, a biological substance having an amino group, a thiol group,a hydroxyl group, carboxyl group, a bromoatomamido group or the like atits terminal can be fixed on the surface of the Si oxide layer of thesubstrate B₀ by using the linker substance.

Further, by using double-chain DNA, proteins, peptides, sugars,RNA-protein complexes, or sugar-protein complexes as the biologicalsubstances, chips for detecting transcription factors that identify aspecific base sequence of double-chain DNA and bond to it, a chip fordetecting peptides, a chip for detecting proteins, a chip for detectingsugars, a chip for digesting proteins or the like can be made.

SECOND EMBODIMENT

Using an SOI wefer (produced by Shin-Etsu Chemical Co., Ltd.) as astarting material, a substrate B₁ having a final structure shown in FIG.9 was made by the process shown in FIGS. 10 to 14.

The specifications of the substrate B₁ were as follows:

Size: 1 cm×1 cm; Shape of bottomed wells: 300 μm in diameter and 20 μmin depth; The bottomed wells were arranged in a grid pattern in thesubstrate, where 9 wells were in one line and 14 wells were in one row.

The silane layer was formed using 5,6-epoxytriethoxysilane, and theoligonucleotide spacer layer was formed by the phosphoramidite methodusing a DNA synthesizing reagent produced by Proligo Japan K. K.

Using this substrate B₁ and applying the phosphoramidite method, a DNAchip was made, where probe DNA having the sequences below wassynthesized in the wells.

1. 3′-ATCTCACACGTCAAATAG-5′ 2. 3′-ATCTCACTCAAATAG-5′ 3.3′-ATCTCACGCAAATAG-5′ 4. 3′-ATCTCACCCAAATAG-5′ 5. 3′-ATCTCACACAAATAG-5′6. 3′-ATCTCACCAAATAG-5′

Using this DNA chip, fluorescence-marked target DNA detection test wasperformed. FIG. 23 is a photograph showing the results.

As seen in FIG. 23, spots corresponding to probe 4 were highest influorescence intensity. From this, it turned out that the sequence ofthe target DNA was 5′-TAGAGTGGGTTTATC-3′.

In each of probes 2, 3 and 4, a base located at the center of thesequence causes a mismatch. In probe 1, the base sequence is too longand causes a mismatch, while in probe 6, the base sequence is too shortand causes a mismatch.

For comparison, a substrate A having a structure shown in FIG. 7 wasmade by performing capping according to the method disclosed in PCTApplication Published Japanese Translation No. 2002-537869.

Using this substrate A, a DNA chip in which probe DNA having theabove-mentioned 6 sequences was fixed was made in the same manner as inthe Example.

Target DNA detection test was performed in the same manner as in theExample. FIG. 24 shows the results.

Also in this DNA chip, spots corresponding to probe 4 were highest influorescence intensity. Hence, also from this DNA chip, it turned outthat the sequence of the target DNA was 5′-TAGAGTGGGTTTATC-3′.

It is to be noted that as seen in FIG. 24, in the DNA chip made from thesubstrate A, the part surrounding each spot was fluorescent-colored,that is, background noise was produced, which made the boundary betweenthe fluorescent-colored spots and the substrate surface unclear.

This comes from the fact that when the wells were filled with resindroplets in the process of making the substrate, the resin ran over thewells to their surrounding parts, so that these surrounding parts didnot undergo capping.

In the spots corresponding to probe 4, a central part of each spot wasweek in fluorescent color. This comes from the fact that too less anamount of resin droplets was supplied to fill the wells. Meanwhile, eachspot as a whole was high in fluorescence intensity. This is because theprobe DNA fixed in these spots was complementary to the target DNA.

Compared with the substrate A, in the case of the substrate B that is anexample of a second embodiment of the present invention, the boundarybetween the fluorescent-colored spots and the substrate surface was veryclear and background noise was virtually not produced.

Further, each fluorescent-colored spot showed an even fluorescenceintensity, and measured data was stable.

THIRD EMBODIMENT

With reference to FIGS. 25 and 26, a substrate for a biochip accordingto a third embodiment of the present invention has a semiconductorsubstrate 15, a first layer 13 having a surface to be hydroxylated anddisposed on the semiconductor substrate 15, and a second layer 11disposed on the first layer 13. The second layer 11 has a plurality ofwells 41 a, 41 b, 41 c, 41 d, 41 e, 41 f, 41 g, 41 h, 41 i, 42 a, 42 b,42 c, 42 d, 42 e, 42 f, 42 g, 42 h, 42 i, 43 a, 43 b, 43 c, 43 d, 43 e,43 f, 43 g, 43 h, 43 i, 44 a, 44 b, 44 c, 44 d, 44 e, 44 f, 44 g, 44 h,44 i, 45 a, 45 b, 45 c, 45 d, 45 e, 45 f, 45 g, 45 h, 45 i, 46 a, 46 b,46 c, 46 d, 46 e, 46 f, 46 g, 46 h, 46 i, 47 a, 47 b, 47 c, 47 d, 47 e,47 f, 47 g, 47 h, 47 i, 48 a, 48 b, 48 c, 48 d, 48 e, 48 f, 48 g, 48 h,48 i, 49 a, 49 b, 49 c, 49 d, 49 e, 49 f, 49 g, 49 h, 49 i. Each of thewells 41 a-49 i reaches to the first layer 13.

Further, the second layer 11 shown in FIG. 25 has a plurality of grooves31 a, 31 b, 31 c, 31 d, 31 e, 31 f, 31 g, 31 h, 31 i, 32 a, 32 b, 32 c,32 d, 32 e, 32 f, 32 g, 32 h, 32 i, 33 a, 33 b, 33 c, 33 d, 33 e, 33 f,33 g, 33 h, 33 i, 34 a, 34 b, 34 c, 34 d, 34 e, 34 f, 34 g, 34 h, 34 i,35 a, 35 b, 35 c, 35 d, 35 e, 35 f, 35 g, 35 h, 35 i, 36 a, 36 b, 36 c,36 d, 36 e, 36 f, 36 g, 36 h, 36 i, 37 a, 37 b, 37 c, 37 d, 37 e, 37 f,37 g, 37 h, 37 i, 38 a, 38 b, 38 c, 38 d, 38 e, 38 f, 38 g, 38 h, 38 i,39 a, 39 b, 39 c, 39 d, 39 e, 39 f, 39 g, 39 h delineated in the secondlayer 11. Each of the grooves 31 a-39 h connects the wells 41 a-41 i, 42a-42 i, 43 a-43 i, 44 a-44 i, 45 a-45 i, 46 a-46 i, 47 a-47 i, 48 a-48i, 49 a-49 i to fill the wells 41 a-49 i with a same solution.

The first layer 13 composed of silicon dioxide (SiO₂). The second layer11 is composed of material different from the first layer 13. Forexample, the second layer 11 is composed of crystal silicon. The firstlayer 13 has higher hydrophilicity than the second layer 11. Therefore,the first layer 13 is easily hydroxylated in comparison with the secondlayer 11.

With reference to FIGS. 26 and 27, the groove 31 a connects the well 41a and the well 41 b. Similarly, each of the other grooves 31 b-31 i, 32a-32 i, 33 a-33 i, 34 a-34 i, 35 a-35 i, 36 a-36 i, 37 a-37 i, 38 a-38i, 39 a-39 h connects the wells 41 b-41 i, 42 a-42 i, 43 a-43 i, 44 a-44i, 45 a-45 i, 46 a-46 i, 47 a-47 i, 48 a-48 i, 49 a-49 i.

In FIG. 26, each of biomolecule layers 91 a, 91 b, 91 c, 91 d, 91 e, 91f, 91 g, 91 h, 91 i are disposed on the surface of the first layer 13exhibited by each of the wells 41 a-41 i. In each of the biomoleculelayers 91 a-91 i, a plurality of probe biomolecules are covalentlylinked to the first layer 13 by forming covalent bonds betweenfunctional groups in the probe biomolecules and hydroxyl (—OH) groups onthe first layer 13. Each of the “probe biomolecules” may involve adeoxyribonucleic acid (DNA) chain, a ribonucleic acid (RNA) chain, apeptide nucleic acid (PNA) chain, or a protein. In a case where theprobe biomolecules are DNA chains, RNA chains, or PNA chains, eachsequence of the probe biomolecules is designed to be complementary toeach of target biomolecules. FIG. 28 depicts the DNA chains in thebiomolecule layer 91 a being covalently linked to the first layer 13.

It should be noted that the third embodiment is not limited to a casewhere each of the biomolecule layers 91 a-91 i is disposed on the firstlayer 13 directly. With reference to FIG. 29, a plurality of silanefilms 81 a, 81 b, 81 c, 81 d, 81 e, 81 f, 81 g, 81 h, 81 i are disposedon the first layer 13 exhibited by each of the wells 41 a-41 i. In eachof the silane films 81 a-81 i, a plurality of silane coupling agentsform a matrix on the first layer 13. With reference to FIG. 30, acovalent bond is formed from the acid-base reaction of a methoxy group(—OCH₃) in each of the silane coupling agents with the hydroxyl (—OH)group on the first layer 13 by acid-base reaction.

For example, a 3-glycidoxypropyltrimethoxysilane, a3-glycidoxypropylmethyldiethoxysilane, a3-glycidoxypropyltriethoxysilane, anN-2(aminoethyl)3-aminopropylmethyldimethoxysilane, anN-2(aminoethyl)3-aminopropyltrimethoxysilane, anN-2(aminoethyl)3-aminopropyltriethoxysilane, a3-aminopropyltrimethoxysilane, and a 3-aminopropyltriethoxysilane can beused for each of the silane coupling agents.

With reference again to FIG. 29, each of biomolecule layers 91 a, 91 b,91 c, 91 d, 91 e, 91 f, 91 g, 91 h, 91 i is disposed on each of thesilane films 81 a-81 i. In boundary between each of the biomoleculelayers 91 a-91 i and each of the silane films 81 a-81 i, an amide bond(—NH—CO—) is formed from the reaction of an active ester introduced toeach of the probe biomolecules with each amino group (—NH₂) of thesilane coupling agents as shown in FIG. 30, for example.

Further, linking each of the silane coupling agents and each of theprobe biomolecules by using a crosslinker is an alternative. Proteinsuch as a receptor, a ligand, an antagonist, an antibody, and an antigencontains the functional group such as an amino group (—NH₂) in a lysine(Lys), a carboxyl group (—COOH) in an aspartic acid (Asp) and a glutamicacid (Glu), a phenol group (—C₆H₄(OH)) in a tyrosine (Tyr), an imidazolegroup (—C₃H₃N₂) in a histidine (His), and a thiol group (—SH) in acystein. Therefore, the crosslinkers that are reactive toward the aminogroups in both ends such as a disuccinimidyl suberate (DSS), abis[sulfosuccinimidyl]suberate (BS³), a dimethyl suberimidate HCl (DMS),a disuccinimidyl glutarate (DSG), a Loman's reagent, a3,3′-dithiobis[sulfosuccinimidyl propionate] (DTSSP), an ethylene glycolbis[succinimidylsuccinate] (EGS) can be used for linking the probebiomolecules and the silane coupling agents. Also, the crosslinkers thatare reactive toward the amino group and the carboxyl group such as a1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) can beused.

Further, the crosslinkers that are reactive toward the amino group andthe thiol group such as a m-maleimidobenzyl-N-hydroxysuccinimide ester(MBS), a succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate(SMCC), a succinimidyl 4-p-malemidophenyl]-buthrate (SMPB), anN-Succinimidyl3-[2-pyridyldithio]propionate (SPDP), anN-[γ-Maleimidobutylody]sulcosuccinimide ester (Sulfo-GMBS), asulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamide]hexanoate(Sulfo-LC-SPDP), an m-maleimidebenzoyl-N-hydroroxysulfo-succinimideester (Sulfo-MBS), a sulfosuccinimidyl4[N-maleimidomethyl]-cyclohexane-1-carboxylate (Sulfo-SMCC), asulfosuccinimidy 4-[p-maleimidophenyl]-butyrate (Sulfo-SMPB) can be usedfor linking the probe biomolecules and the silane coupling agents.

In FIG. 31, each amino group of the silane coupling agents and eachamino group of the antibodies 95 a, 95 b, 95 c are coupled with the DSS.

Each sectional view of the other wells 42 a-42 i, 43 a-43 i, 44 a-44 i,45 a-45 i, 46 a-46 i, 47 a-47 i, 48 a-48 i, 49 a-49 i shown in FIG. 25is similar to FIG. 26 or FIG. 29.

It should be noted that a silica glass also can be used for the firstlayer 13 shown in FIGS. 26 and 29. In this case, the semiconductorsubstrate 15 can be eliminated. Also, a resin such aspolytetrafluoroethylene and insoluble epoxy resist also can be used forthe second layer 11.

The surface of the substrate for the biochip shown in FIGS. 25-31 iscovered by a cover plate 25 shown in FIGS. 32 and 33. The cover plate 25has openings 27, 28. The cover plate 25 is composed of fused silica,acrylic resin, and polycarbonate, for example. FIG. 34 depicts asectional view in the case where the cover plate 25 is disposed on thesubstrate for the biochip shown in FIG. 25.

When a hybridization test is performed by the substrate for the biochip,sample solution containing a plurality of sample biomolecules labeledwith fluorescent dye such as Cy3, or Cy5 are prepared. Such samplesolution may be dispensed into the opening 27. By sucking air from theopening 28, each of the wells 41 a-41 i and each of the grooves 31 a-31i shown in FIG. 34 are filled with the sample solution in order.Further, each of the other wells 42 a-42 i, 43 a-43 i, 44 a-44 i, 45a-45 i, 46 a-46 i, 47 a-47 i, 48 a-48 i, 49 a-49 i and each of the othergrooves 32 a-32 i, 33 a-33 i, 34 a-34 i, 35 a-35 i, 36 a-36 i, 37 a-37i, 38 a-38 i, 39 a-39 h shown in FIG. 25 are also filled with the samplesolution in order.

In the case where the sample biomolecules contain a plurality of targetbiomolecules that bond complementarily with the probe biomolecules inthe biomolecule layers 91 a-91 i, the target biomolecules may be trappedin the biomolecule layers 91 a-91 i. After sucking the sample solution,the wells 41 a-41 i, 42 a-42 i, 43 a-43 i, 44 a-44 i, 45 a-45 i, 46 a-46i, 47 a-47 i, 48 a-48 i, 49 a-49 i and the grooves 31 a-31 i, 32 a-32 i,33 a-33 i, 34 a-34 i, 35 a-35 i, 36 a-36 i, 37 a-37 i, 38 a-38 i, 39a-39 h are rinsed out by buffer. Thereafter, by observing fluorescentreactions, it is possible to examine whether the sample biomoleculescontain the target biomolecules or not. The fluorescent reactions arevisible to the naked eye in the case where each diameter of the wells 41a-41 i, 42 a-42 i, 43 a-43 i, 44 a-44 i, 45 a-45 i, 46 a-46 i, 47 a-47i, 48 a-48 i, 49 a-49 i is larger than 600 μm.

As described above, since the substrate for the biochip shown in FIGS.25-34 has the wells 41 a-41 i, 42 a-42 i, 43 a-43 i, 44 a-44 i, 45 a-45i, 46 a-46 i, 47 a-47 i, 48 a-48 i, 49 a-49 i and the grooves 31 a-31 i,32 a-32 i, 33 a-33 i, 34 a-34 i, 35 a-35 i, 36 a-36 i, 37 a-37 i, 38a-38 i, 39 a-39 h delineated in the second layer 11, it is possible toeliminate precise alignment of the cover plate 25 on the substrate forthe biochip. Further, required sample solution volume is equal to thetotal volume of the wells 41 a-41 i, 42 a-421, 43 a-43 i, 44 a-44 i, 45a-45 i, 46 a-46 i, 47 a-47 i, 48 a-48 i, 49 a-49 i and the grooves 31a-31 i, 32 a-32 i, 33 a-33 i, 34 a-34 i, 35 a-35 i, 36 a-36 i, 37 a-37i, 38 a-38 i, 39 a-39 h. Therefore, it is possible to reduce the samplesolution volume. Also, the grooves 31 a-31 i, 32 a-32 i, 33 a-33 i, 34a-34 i, 35 a-35 i, 36 a-36 i, 37 a-37 i, 38 a-38 i, 39 a-39 h eliminatethe need to fill each of the wells 41 a-41 i, 42 a-421, 43 a-43 i, 44a-44 i, 45 a-45 i, 46 a-46 i, 47 a-47 i, 48 a-48 i, 49 a-49 i with thesample solution by spotting device. Therefore, it is possible to shortenthe hybridization test period.

With reference next to FIGS. 35-56, a method for manufacturing thesubstrate for the biochip according to the third embodiment isdescribed.

In FIG. 35, an SOI (Silicon On Insulator) substrate having thesemiconductor substrate 15, the first layer 13 composed of SiO₂ anddisposed on the semiconductor substrate 15, the second layer 11 composedof the crystal silicon and disposed on the first layer 13, and a nativeoxide film 12 disposed on the second layer 11 is prepared. Then, aresist 21 is coated on the native oxide film 12 by a spin coater asshown in FIG. 36.

The parts of the resist 21 are selectively etched by using aphotolithography process. Accordingly, a plurality of openings 51 a, 51b, 51 c, 51 d, 51 e, 51 f, 51 g, 51 h, 51 i are formed in the resist 21as shown in FIG. 37. Thereafter, the native oxide film 12 exhibited bythe openings 51 a-51 i and the parts of the second layer 11 areselectively etched. Accordingly, the grooves 31 a-31 i, 32 a-32 i, 33a-33 i, 34 a-34 i, 35 a-35 i, 36 a-36 i, 37 a-37 i, 38 a-38 i, 39 a-39 hare delineated in the second layer 11 as shown in FIGS. 38 and 39.

In FIG. 40, a resist 22 is coated on the native oxide film 12 by thespin coater. Thereafter, the parts of the resist 22 are selectivelyetched by using the photolithography process. Accordingly, a pluralityof openings 61 a, 61 b, 61 c, 61 d, 61 e, 61 f, 61 g, 61 h, 61 i areformed in the resist 22 as shown in FIG. 41. Thereafter, the nativeoxide film 12 exhibited by the openings 61 a-61 i and the parts of thesecond layer 11 are selectively etched until the first layer 13 isexhibited. Consequently, the wells 41 a-41 i are delineated in thesecond layer 11 as shown in FIG. 42.

The SOI substrate is dipped into a stirred sodium hydrate solution fortwo hours at room temperature. The sodium hydrate solution is made bymixing 98 g of sodium hydrate, 294 ml of distilled water, and 392 ml ofethanol. By dipping the SOI substrate into the sodium hydrate solution,the native oxide film 12 on the second layer 11 is removed as shown inFIG. 43. Further, the parts of the surface of the first layer 13exhibited by the wells 41 a-41 i are hydroxylated. Therefore, aplurality of hydroxyl (—OH) groups are introduced on the parts of thesurface of the first layer 13 as shown in FIG. 44.

The silane coupling agent having the epoxy group such as the3-glycidoxypropylmethyldiethoxysilane, the3-glycidoxypropyltriethoxysilane, or the silane coupling agent havingthe amino group such as the N-2(aminoethyl)3-aminopropyltriethoxysilane,the 3-aminopropyltrimethoxysilane, and the 3-aminopropyltriethoxysilaneis dispensed on the parts of the surface of the first layer 13 exhibitedby the wells 41 a-41 i shown in FIG. 43. Consequently, the silane films81 a, 81 b, 81 c, 81 d, 81 e, 81 f, 81 g, 81 h, 81 i are formed on thefirst layer 13 as shown in FIG. 45. When the3-aminopropyltrimethoxysilane is dropped on the first layer 13, aplurality of the amino (—NH₂) groups are introduced on the surface ofthe first layer 13 as shown in FIG. 46. Thereafter, remaining freehydroxyl (—OH) groups on the first layer 13 are capped to preventparticipating in the rest of synthesis reactions. For example, theremaining free hydroxyl (—OH) groups are acetylated by acetic anhydrideand 1-methyl imidazole (tetrahydrofuran solution).

A solution containing first nucleosides having active esters isprepared. Each of the first nucleosides is blocked at the 5′-hydroxylwith a dimethoxytrityl (DMTr) group. In each base of the firstnucleosides, an amino group of an adenine or a cytosine is protectedwith a benzoyl group, and an amino group of a guanine is protected withan isobutyl group as shown in FIG. 47. The solution containing the firstnucleosides is dispensed onto the silane film 81 a shown in FIG. 46.Accordingly, each active ester of the first nucleosides reacts with eachamino group of the silane coupling agents to form an amide bond(—NH—CO—) as shown in FIG. 48. Thereafter, each DMTr of the firstnucleosides is removed by 3% trichloroacetic acid in dichloromethane(DCM). In FIG. 49, the 5′ hydroxyl (—OH) group is now the only reactivegroup on each of the first nucleosides.

A solution containing tetrazoles and a plurality of phosphoramiditesthat are derivatives of second nucleosides shown in FIG. 50 is dispensedonto the silane films 81 a-81 i. In FIG. 51, each active 5′ hydroxyl(—OH) group of the first nucleosides and each N,N-di-isopropylamine ofthe second nucleosides form an unstable phosphite linkage by acondensation reaction. Thereafter, the unbounded, active 5′-hydroxyl(—OH) groups of the first nucleosides are capped with protective groups.For example, the 5′-hydroxyl (—OH) groups are acetylated by addingacetic anhydride and 1-methylimidazole onto the first layer 13.

To stabilize the unstable phosphite linkage, a solution of dilute iodinein water, pyridine, and tetrahydrofuran is added onto the first layer13. In FIG. 52, the unstable phosphite linkage is oxidized to form amuch more stable phosphate linkage. Thereafter, each DMTr of the secondnucleosides is removed and the condensation reactions are repeated untilchain elongation is complete as shown in FIG. 53.

In FIG. 54, each of cyanoethyl groups protecting the phosphate linkageis cleaved by concentrated ammonia. Also, each of heterocyclicprotection groups such as the benzoyl group and the isobutyl group shownin FIG. 47 is cleaved by the concentrated ammonia and each ofheterocyclic primary amines is deprotected as shown in FIG. 55. Further,each DMTr on the very last bases is cleaved as shown in FIG. 56. Theprocesses explained with FIGS. 48-56 are also performed on the silanefilms 81 b-81 i shown in FIG. 45 and the biomolecule layers 91 a-91 iare formed as shown in FIG. 29.

As described above, the method for manufacturing the substrate for thebiochip according to the third embodiment involves treatment of the SOIsubstrate with the sodium hydrate solution. Therefore, the hydroxyl(—OH) groups are only introduced on the surface of the first layer 13.Also, in the case where the second layer 11 is composed of Si, thethickness of the native oxide film 12 is usually 2 nm or below. Thesodium hydrate solution effectively removes such native oxide film 12 atroom temperature and for two hours. Consequently, the silane couplingagents added later only react with the hydroxyl (—OH) groups on thefirst layer 13 and may not bind to the surface of the second layer 11.In earlier methods, the silane coupling agents may react with theremaining native oxide film 12. Therefore, the target biomoleculeslabeled with the fluorescent dyes may also be immobilized on the nativeoxide film. Such immobilized target biomolecules on the native oxidefilm cause background noise. Especially in micro analysis, it has beendesired to develop the method to immobilize the target biomolecules onlyon the bottoms of the wells 1 a-41 i, 41 b-41 i, 42 a-42 i, 43 a-43 i,44 a-44 i, 45 a-45 i, 46 a-46 i, 47 a-47 i, 48 a-48 i, 49 a-49 i andprevent the target biomolecules from binding to the surface of thesecond layer 11, since such method improves contrast in the fluorescenceanalysis. The method for manufacturing the substrate for the biochip mayserve such demand since the hydroxyl (—OH) groups are only introduced onthe surface of the first layer 13 and the native oxide film 12 isremoved from the surface of the second layer 11. Therefore, it ispossible to manufacture the substrate for the biochip that may excludethe background noise.

FIG. 57( a) shows an example microscope image when the wells 41 a-42 i,42 a-42 i shown in FIG. 25 are exposed to sample solution containingsample DNA labeled with the fluorescent dye and having a sequence thatis complementary to a sequence of the DNA immobilized in the biomoleculelayer 91 a shown in FIG. 28. FIG. 57( b) shows an example microscopeimage when the earlier substrate for the biochip shown in FIGS. 2-7 areexposed to the sample DNA labeled with the fluorescent dye and having asequence that is complementary to a sequence on the substrate. In theearlier substrate, the sample DNA binds to not only the bottom of thewells but also on other areas. Also, contaminations are detected on thebottom of the wells. However, the substrate for the biochip according tothe third embodiment makes it possible to hybridize the target DNA onlyon the bottoms of the wells as shown in FIG. 57( a). Also, theuniformity of the fluorescent on the wells is realized.

In FIGS. 37-42, the grooves 31 a-31 i, 32 a-32 i, 33 a-33 i, 34 a-34 i,35 a-35 i, 36 a-36 i, 37 a-37 i, 38 a-38 i, 39 a-39 h are delineated inthe second layer 11 in advance. Then the wells 41 a-41 i, 41 b-41 i, 42a-42 i, 43 a-43 i, 44 a-44 i, 45 a-45 i, 46 a-46 i, 47 a-47 i, 48 a-48i, 49 a-49 i are delineated in the second layer 11. Contrary, it shouldbe noted that forming the wells 41 a-41 i, 41 b-41 i, 42 a-42 i, 43 a-43i, 44 a-44 i, 45 a-45 i, 46 a-46 i, 47 a-47 i, 48 a-48 i, 49 a-49 i inadvance and then forming the grooves 31 a-31 i, 32 a-32 i, 33 a-33 i, 34a-34 i, 35 a-35 i, 36 a-36 i, 37 a-37 i, 38 a-38 i, 39 a-39 h are alsoalternative. However, since each depth of the grooves 31 a-31 i, 32 a-32i, 33 a-33 i, 34 a-34 i, 35 a-35 i, 36 a-36 i, 37 a-37 i, 38 a-38 i, 39a-39 h is shallower than each depth of the wells 41 a-41 i, 41 b-41 i,42 a-42 i, 43 a-43 i, 44 a-44 i, 45 a-45 i, 46 a-46 i, 47 a-47 i, 48a-48 i, 49 a-49 i, forming the grooves 31 a-31 i, 32 a-32 i, 33 a-33 i,34 a-34 i, 35 a-35 i, 36 a-36 i, 37 a-37 i, 38 a-38 i, 39 a-39 h inadvance makes it easier to coat the resist on the second layer 11uniformly.

Also, in FIGS. 45-56, the biomolecule layers 91 a-91 i are formed afterthe silane films 81 a-81 i on the first layer 13. However, attaching thebiomolecules to the hydroxyl (—OH) groups on the first layer 13 andforming the covalent bonds without the silane films 81 a-81 i are alsoalternative.

Further, though the SOI substrate is prepared in FIG. 35, manufacturingthe substrate for the biochip by using a laminate sheet consisting of aglass substrate and a polytetrafluoroethylene substrate is alsoavailable. In this case, the glass substrate is used as the first layer13 and the polytetrafluoroethylene substrate is used as the second layer11.

FIRST MODIFICATION OF THE THIRD EMBODIMENT

The method for manufacturing the substrate for the biochip is notlimited to the third embodiment described above. With reference now toFIGS. 58-62, the method for manufacturing the substrate for the biochipusing the epoxy negative resist is describe.

In FIG. 58, a first resist 111 composed of the epoxy negativephoto-resist is coated on the first layer composed of the glass by thespin coater, for example. Thereafter, a first photo mask 40 is preparedin FIG. 59. The first photo mask 40 has a plurality of light shieldpatterns 140 a, 140 b, 140 c. The light shield patterns 140 a-140 ccorrespond to the shape of the wells to be fabricated in the firstresist 111. Then, light is shone over the first photo mask 40 and theportions of the first resist 111 are exposed to the light.

In FIG. 60, a second resist 211 composed of the epoxy negativephoto-resist is coated on the first resist 111 by the spin coater.Accordingly, the first resist 111 and the second resist 211 forms thesecond layer 11. In FIG. 61, a second photo mask 50 is prepared. Thesecond photo mask 50 has a light shield pattern 150. The light shieldpattern 150 corresponds to the shape of the wells and the grooves formedin the second layer 11. Then, the light is shone over the second photomask 50 and the portions of the second resist 211 are exposed to thelight.

The exposed first resist 111 and the exposed second resist 211 arebaked. It should be noted that the post exposure bake renders the firstresist 111 and the second resist 211 insoluble in buffer. Thereafter,the first resist 111 and the second resist 211 are developed with analkaline developer. Consequently, the wells 41 a-41 i and the grooves 31a-31 i are formed in the second layer 11 as shown in FIG. 62.Subsequently, the method explained with FIGS. 44-56 is carried out andthe method for manufacturing the substrate for the biochip is completed.

SECOND MODIFICATION OF THE THIRD EMBODIMENT

With reference now to FIGS. 63 and 64, a substrate for a biochipaccording to a second modification of the third embodiment differs withthe substrate shown in FIGS. 25 and 26 in the absence of the grooves 31a-31 i, 32 a-32 i, 33 a-33 i, 34 a-34 i, 35 a-35 i, 36 a-36 i, 37 a-37i, 38 a-38 i, 39 a-39 h delineated in the second layer 11. Withreference next to FIGS. 65 and 66, a cover plate 125 according to asecond modification of the third embodiment differs with the cover plate25 shown in FIGS. 32 and 33 in the presence of a groove 300 shown inFIGS. 65 and 66. In the case where the cover plate 125 is disposed onthe substrate shown in FIGS. 63 and 64, the groove 300 connects thewells 41 a-41 i, 41 b-41 i, 42 a-42 i, 43 a-43 i, 44 a-44 i, 45 a-45 i,46 a-46 i, 47 a-47 i, 48 a-48 i, 49 a-49 i.

FIG. 67 depicts a sectional view in the case where the cover plate 25shown in FIG. 65 is disposed on the substrate for the biochip shown inFIG. 63. The sample solution dispensed into the opening 27 may fill thewell 41 a. Thereafter, the sample solution flows the groove 300 andfills the wells 41 b-41 i in order. Therefore, as similar to the thirdembodiment, the substrate for the biochip shown in FIG. 63 and the coverplate 25 shown in FIG. 65 makes it possible to reduce the volume of thesample solution.

This application is based upon and claims the benefit of priority fromprior European Patent Application 04291742.7 filed on Jul. 8, 2004; theentire contents of which are incorporated by reference herein.

INDUSTRIAL APPLICABILITY

As is clear from the description of the substrate for synthesizing probeDNA, in the substrate for biochips according to the present invention,the boundary between the reaction region for synthesizing biologicalsubstances and the non-reaction region is quite definite. Hence, abiochip made using this substrate makes it possible to detectto-be-detected biological substances stably and with high accuracy.

Further, in the case of the substrate according to the invention, sitesforming the reaction region are formed by photolithography and etchingin the process of making the substrate. This increases the freedom offormation and enables fine patterns and therefore high-density formationof the reaction region. Further, since the making process does not needto include the steps of filling the bottomed wells with resin dropletsand then performing inactivation treatment, the overall production costsdecrease.

As a substrate used for making DNA chips, RNA chips, protein chips,antibody chips, sugar chain fixing chips, bioreactors and the like, thissubstrate for biochips has a large industrial value.

1. A substrate for a biochip comprising a substrate surface having areaction region capable of reacting with biological substances and anon-reaction region not reacting with the biological substances; sunkenbottomed wells formed in said substrate surface; and a layer of amaterial capable of reacting with the biological substances having asurface exposed only at the bottoms of the bottomed wells, said exposedsurface forming said reaction region.
 2. The substrate of claim 1,wherein the substrate includes a plate on which said layer of thematerial not reacting with the biological substances is formed on saidlayer of the material capable of reacting with the biologicalsubstances, and holes functioning as said bottomed wells are formed insaid layer of the material not reacting with the biological substancesin the manner that the surface of said layer of the material capable ofreacting with the biological substances is exposed in the holes.
 3. Thesubstrate of claim 1, wherein said material capable of reacting with thebiological substances is an Si oxide.
 4. The substrate for biochips ofclaim 1, wherein the substrate includes an Si plate on a surface ofwhich an Si oxide layer is formed, at least a layer made of a materialnot reacting with the biological substances is formed on the surface ofsaid Si oxide layer, and holes functioning as said bottomed wells areformed across the thickness of said layer up to the surface of said Sioxide layer.
 5. The substrate of claim 1, wherein said substrateincludes a glass plate on which at least a layer made of a material notreacting with the biological substances is formed, and holes functioningas said bottomed wells are formed across the thickness of said layer upto the surface of said glass plate.
 6. The substrate of claim 1, whereinsaid substrate includes a plate the whole of which is made of a materialnot reacting with the biological substances and which has a layer of amaterial capable of reacting with the biological substances formedinside, and holes functioning as said bottomed wells are formed in saidplate in the manner that the surface of said layer of the materialcapable of reacting with the biological substances is exposed in theholes.
 7. The substrate of claims 1, wherein the surface of said Sioxide layer is silanized.
 8. A substrate for a biochip comprising: afirst layer having a surface to be hydroxylated; and a second layerdisposed on the first layer, the second layer having a plurality ofwells reaching to the first layer and a plurality of grooves configuredto fill the wells with a same solution.
 9. The substrate of claim 8,wherein the first layer has higher hydrophilicity than the second layer.10. The substrate of claim 8, wherein the first layer contains a silicondioxide.
 11. The substrate of claim 8, wherein the second layer containsa crystal silicon.
 12. The substrate of claim 8, wherein the secondlayer contains a resin.
 13. The substrate of claim 8, further comprisinga biomolecule layer having a plurality of probe biomolecules linked to aplurality of hydroxyl groups to be introduced on the first layer. 14.The substrate of claim 13, wherein each of the probe biomolecules andeach of the hydroxyl groups are linked by a silane coupling agent. 15.The substrate of claim 13, wherein each of the probe biomoleculescontains a deoxyribonucleic acid.
 16. The substrate of claim 13, whereineach of the probe biomolecules contains a ribonucleic acid.
 17. Thesubstrate of claim 13, wherein each of the probe biomolecules contains apeptide.
 18. The substrate of claim 13, wherein each of the probebiomolecules contains a protein.
 19. A method for manufacturing asubstrate for a biochip comprising: etching portions of a second layerdisposed on a first layer and exhibiting the first layer; and dippingthe first layer into a sodium hydrate solution to introduce a pluralityof hydroxyl groups on the first layer.
 20. The method of claim 19,further comprising etching portions of the second layer to form aplurality of grooves.
 21. The method of claim 19, further comprisingdipping the second layer into the sodium hydrate solution to remove anative oxide film on the second layer.
 22. The method of claim 19,further comprising linking each of a plurality of probe biomolecules toeach of the hydroxyl groups.
 23. The method of claim 22, wherein thelinking each of the probe biomolecules to each of the hydroxyl groupsfurther comprises bonding a silane coupling agent to each of thehydroxyl groups.